Studies of molecular events inside of cells have provided novel and important insights into many questions in physiology and pathology: How do cellular motors work? How does a proton pump generate ATP? How is cargo secreted from cells? How do viruses enter a cell and, once in, how do they assemble. This application has three aims that will significantly advance ability to follow single molecular events in living cells. The first specific aim develops the technology for loading probes into cells. The technique is efficient and directly delivers probe into the cytosol rather than into the endocytic pathway. Of particular interest for us is the ability to deliver inteins. The second specific aim will generate inteins that will function inside of cells at physiological temperatures at faster rates and higher efficiencies. The third specific aim uses two biological questions to test the strengths and limitations of the results from the first three specific aims. The two questions relate to transport in and out of the nucleus: Can we follow the conformational changes of proteins in single nuclear pores and can we follow the transport of single molecules as they interact with the nuclear pore components and move through the pore. These questions are significant for understanding a fundamental process in cell biology. They will also provide insight as to further modifications that are needed in our technology for studying single molecular events in living cells. PUBLIC HEALTH RELEVANCE: Optical imaging has contributed very significant advances to our understanding of biology in the last few years. This is the consequence of a number of advantages of imaging: Imaging allows us to study biological systems that are still alive;Imaging allows us to study individual molecules, individual cells rather than the average behavior;Imaging allows us to study many different size scales from single molecules to whole individuals. Often a defect that occurs at the level of a single molecule represents itself as pathology at the level of the whole organism. Imaging allows us to follow the disease from the single molecule, to the molecular machine, to the whole cell, to the whole organ, to the individual patient. A major limitation of optical imaging has been the capability to get probes into cells to specifically and selectively label individual molecules. This application will contribute to our ability to study individual molecular events in the cell by giving us new methods of putting our probes into cells, without disturbing the cells and new methods of labeling cellular components with minimal, often no, effect on the cell.